Streptokinase, an efficient activator of fibrinolysis, is routinely utilized in clinical medicine for thrombolytic therapy for the treatment of diverse circulatory disorders, e.g., pulmonary thrombo-embolism, deep vein thrombosis, and myocardial infarction. It exerts its fibrinolytic effects through activation of an inert blood Zymogen, Plasminogen (PG), an active serine protease, Plasmin (PN), which attacks on fibrin to degrade it into soluble degradation products. It has been clearly demonstrated, particularly in the case of myocardial infarction, that in the actual management of the disease, streptokinase is as efficacious as its more expensive clot-dissolving alternatives, such as Urokinase (UK) and tissue-Plasminogen activator (tPA). Its utility in thrombolytic therapy is well established. Reference maybe made to the publications of Paques, E. P., 1986, Haemostasis, Vol. 16, Suppl. 3, 21; ISIS-3 (Third International Study of Infarct Survival: A randomized comparison of streptokinase vs. tissue plasminogen activator vs. anistreplase and of asprin plus heparin alone among 41,299 cases of suspected acute myocardial infarction) Collaborative Group, 1992, Lancet 339,753.
Streptokinase is a single chain 47-kD protein, consisting of 414 amino acid resides (reference may be made in the context of the biochemical properties of Streptokinase to the review article by Castellino, F. J., 1981, Chem Rev. 81, 431). It is naturally produced and secreted by various strains of hemolytic streptococci along with several other unwanted toxic products, e.g. deoxyribonucleases, streptolysin or hyaluronidase and proteases, which makes the process of purifying the desired protein difficult. On the other hand it has not yet been possible to obtain genetically improved strains from these hosts due to the lack of a developed methodology for the gene transfer. Considering its therapeutic applicability and clinical implications in thrombolytic therapy, attempts have been made in the past to search for an alternative source of Streptokinase production through recombinant routes. Reference is made to the publication of Malke and Ferretti, Proceedings of the National Academy of Sciences, USA, Vol. 81, p351, 1984; Hagenson et al., 1989, Enzyme and Microbial Technology, Vol 11, 650; Estrada et al., 1992, Biotechnology Vol 10, 1138; Reference may also be made to U.S. Pat. Nos. 5,296,366, 5,240,845, 4,764,469, 2,043,953, Japanese Patent Number 2020828, European Patent No. 489201, and Cuban Patent No. 90. In the work reported in aforesaid publications and patents, the gene encoding for SK has been isolated from its natural host, Streptococcus and cloned into various heterologous hosts, e.g., E. Coli, Bacillus and yeasts.
In order to improve the yield of Streptokinase, in particular, the gene which determines streptokinase C, A and G (References may be made to the publications of Huang et al 1989, Molecular Microbiology Vol 2(3), 197; and Estrada et al, 1992, Biotechnology Vol 10, 1138;) were cloned and expressed in E. coli as well as Streptococcus sanguis (Reference may be made to the publication of Malke et al., 1984, Molecular and General Genetics Vol 1%, 360). In both the cases, protein levels of 0.64 mg/l and 40 μg/l, respectively, were obtained. In the case of E. coli, 94% of the protein recovered was in the periplasmic space and 6% in the cytosol, whereas, in S. sanguis all the enzyme was found extracellularly. Moreover, many clones producing streptokinase were very unstable owing to some lethal activity of the gene product or protein secretion. More recently, intracellular production of streptokinase has been reported in E. Coli (Reference may be made to the publication of Xue-Wu Zhang et al, 1999, Enzyme and Microbial Technology, Vol 24,647) where 300–400 mg/ml SK protein has been obtained from one liter cell culture of E. coli using a rich and complex cell growth medium.
More often recombinant E. coli strain, developed for the overproduction of streptokinase, exhibited low cell density and biomass which affected the overall yield of the protein product. Production of recombinant streptokinase in the methylotrophic yeast, Pichia pastoris, has been attempted by Philips Petroleum Company (Reference may be made to the publication of Hagenson et al., 1989, Enzyme and Microbial Technology, Vol 11, 650) and Cuban group (Reference may be made to the publication of Estrada et al., 1992, Biotechnology Vol 10, 1138; and Cuban Patent Number 90), where 1.8 mg/l and 1.0 g/l, respectively, of SK has been obtained using continuous fermentation and rich growth medium comprising of complex and expensive ingredients. Using a genetically engineered strain of E. coli 645 mg/l of SK production (Reference may be made to the publication of Xue-Wu Zhang et al, 1999, Enzyme and Microbial Technology, Vol. 24, 647) has been reported under fermentative condition using a rich growth medium.